1. Field of the Invention:
This invention relates to a method for the detection and measurement of carboxylic acid ester hydrolases.
2. Description of the Prior Art:
There are three known methods that may be undertaken for the measurement of carboxylic ester hydrolases. They are as follows:
1. Turbidimetric Methods. These methods are designed to measure esterases which act on insoluble esters present in an emulsified form (aided by certain emulsifiers) of sufficient dilution at the time of assay so that optical measurements can be made thereon. The principle of operation lies in the clearing effect that the products of hydrolysis, fatty acids and partial glycerides have on the turbidity of the assay solution. The most common ester used in this type of method has been glycerol trioleate either in its purified form or as olive oil. Since the clearing effect of the fatty acids depends on their ionization, these methods are only applicable in the alkaline pH range. Also, due to design differences in the light paths of different spectrophotometers what may be an acceptable wavelength in one instrument for turbidity measurements is not necessarily the best wavelength in all instruments (Vogel and Zieve, Clinical Chemistry, 9, 168-181 [1963]). Further, turbidimetric methods are relatively insensitive and do not show good linearity of measured activity with enzyme content, especially at high enzyme levels.
2. Measurement of Liberated Fatty Acids. The fatty acids produced after hydrolysis may be measured by a number of methods. They may be titrated after extraction, or they may be continuously titrated during the course of hydrolysis. The latter method allows a kinetic assay of an ester hydrolase to be made but is limited to the alkaline pH range and requires a special recording titrator. Color changes of an acid-base indicator may be measured as the hydrolysis progresses, theoretically yielding a very sensitive assay. However, to be applicable to a wide number of ester hydrolases with different pH optima a number of indicators are required, and it is necessary to match their pK's to the particular hydrolase to be measured as well as to the pK of any buffer present in the solution so as to obtain zero order kinetics. The sensitivity of this type of method is inversely proportional to the amount of buffer present. The liberated fatty acids may, also, be determined by a first conversion to their copper salts and subsequently measured colorimetrically. The most sensitive method involves the use of radioactive esters labeled in the acid portion. The liberated fatty acid, after separation from the unhydrolyzed ester, is counted in a suitable scintillation counter. This type of method however is very time consuming and expensive.
3. Measurement of Liberated Alcohol. Certain esters of phenols are used in this technique and the free phenolic product of hydrolysis is measured colorimetrically. This type of method allows continuous monitoring of the reaction only in the pH range in which the phenol is colored. As an extension of this method and a method of greater sensitivity is that of fluorometric analysis after coupling the liberated phenol with an azo dye. However the specificity of the phenol esters for certain hydrolases is questionable, especially the water soluble phenol esters to triglyceride lipase such as found in the pancreas. Also, pancreatic lipase has a very low specific activity even for water insoluble phenol esters. Rather than phenolic esters, other fluorometric methods utilize carboxylic acid esters of alcohols such as .beta.-naphthol, fluorescein, or 4-methyl-umbelliferone which fluoresce after hydrolysis. These esters are, however, poor substrates for pancreatic lipase. Vinyl esters have also been used to measure hydrolases (Brockerhoff, H., Biochimica et Biophysica Acta, 212, 92 [1970] and Brockerhoff, H. et al, Analytical Biochemistry, 37, 26-31 [1970]). With these esters the OH-containing moiety is not measured, but, its isomerization product, acetaldehyde. In the Brockerhoff technique, the vinyl ester is emulsified, thus, precluding any possible optical measurements on the reaction as it progresses. Aliquots of the reaction mixture containing acetal dehyde are coupled to 3-methyl-2-benzothiazolone hydrazone, thus forming a colored product which is determined colorimetrically at 666nm. However, the vinyl oleate which is used as a substrate is only 29.4% as effective as glycerol trioleate under the same conditions using porcine pancreatic lipase for these measurements. However, vinyl oleate is a much better substrate than the phenolic esters or the esters of the fluorescent alcohols. Significantly, Brockerhoff notes that the kinetics of his method are not linear above an absorbance of about 0.6. The Brockerhoff method, although an improvement over many of the prior art methodologies, is one that must be carried out manually and requires the preparation of several different solutions as well as a great amount of technician time.
Almost all of the methods noted above involve the use of relatively unstable reagent mixtures, especially those which require any kind of an emulsion of a water insoluble substrate. The methods which do use water soluble substrates either have an extremely low specific activity toward triglyceride lipases, or are subject to interference from ester hydrolases which act on water soluble substrates, or both. Triglyceride lipases may, also, interfere with methodologies designed to measure ester hydrolases of water soluble substrates, since the substrate specificities of many triglyceride lipases include water soluble ones as well. A common example of multiple enzyme systems of this type is blood serum or plasma, which may contain pancreatic lipase from an inflamed pancreas as well as a liver esterase. A method designed to differentiate lipase activity from that of the esterase must be highly specific for the former in order to be a reliable diagnostic tool for the detection of pancreatic inflamations. Such reagent solution must be highly sensitive to detect low levels of enzyme activity, stable upon storage for long periods of time, capable of being assayed very quickly after addition of the ester hydrolase (preferably using continuous monitoring techniques to provide for a kinetic assay), free from turbidity effects during the reaction, and economically feasible for manufacturing. A need therefore, exists for a reagent solution which displays high sensitivity, good clarity and storage stability and is capable of quickly measuring a particular ester hydrolase, e.g., triglyceride lipase, even in the presence of other types of ester hydrolases which may have different substrate specificities or other properties.